当前位置: 首页 > 医学版 > 期刊论文 > 基础医学 > 感染与免疫杂志 > 2006年 > 第1期 > 正文
编号:11254926
A 28-Regulated Nonflagella Gene Contributes to Virulence of Campylobacter jejuni 81-176
     Enteric Diseases Department, Naval Medial Research Center, Silver Spring, Maryland 20910

    Food and Drug Administration MOD1, Beltsville, Maryland

    ABSTRACT

    A Campylobacter jejuni 81-176 mutant in Cj0977 was fully motile but reduced >3 logs compared to the parent in invasion of intestinal epithelial cells in vitro. The mutant was also attenuated in a ferret diarrheal disease model. Expression of Cj0977 protein was dependent on a minimal flagella structure.

    TEXT

    The motility imparted by the single polar flagella of Campylobacter jejuni is critical for intestinal colonization and for invasion into intestinal epithelial cells (IEC) in vitro (2, 4, 10, 22, 24, 26, 32, 34, 36). C. jejuni strains appear to use the flagella structure as a type III secretory organelle in the absence of specialized secretion systems for virulence factors. Thus, C. jejuni secretes a set of so-called Cia proteins upon coculture with IECs (19, 20, 29). Mutation of ciaB resulted in loss of secretion of all Cia proteins and reduced invasion of IECs (19, 20, 29). Although the Cia proteins are synthesized in nonmotile mutants, secretion required a minimum flagella filament structure (20). A second protein, FlaC, which shows sequence similarity to flagellin but which is not required for motility or flagella biogenesis, is also secreted through the flagella apparatus (31). FlaC, which is expressed and secreted independently of CiaB, binds to HEp2 cells in vitro and modulates invasion (31).

    A recent microarray study by Carillo et al. (5) reported up-regulation of several 28 and 54 nonflagella genes in a more virulent variant of the genome strain NCTC 11168 (28). These same genes were also down-regulated in two flagella mutants of NCTC 11168. We have confirmed that many of these same genes are also down-regulated in flagella mutants in C. jejuni 81-176 (S. Goon, C. P. Ewing, and P. Guerry, unpublished data), and here we further characterize one of these 28-regulated genes, Cj0977.

    The Cj0977 proteins (Mr, 21.2 kDa; pI 4.8) from 81-176 and NCTC 11168 were 98% identical. The Cj0977 protein lacks a leader sequence or transmembrane domains and is predicted to be cytoplasmic. The protein is conserved within the Proteobacteria and shares some similarity with initiation factor eIF-2B (GenBank accession no. NP_214448). A Cj0977::cat mutant in C. jejuni 81-176 was fully motile (Fig. 1A) and produced a normal flagella filament (Fig. 1B). Purified flagellin from the mutant displayed an isoelectric focusing (IEF) pattern identical to that of 81-176 (Fig. 1C), suggesting that Cj0977 is not involved in flagellin glycosylation (7, 9, 23, 33). There was no difference in growth kinetics of the mutant and the parent 81-176 in Mueller-Hinton (MH) broth, nor were there any changes in either capsular polysaccharide by immunoblot or lipooligosaccharide mobility on silver-stained gels (data not shown).

    The Cj0977 mutant and a complement in which the wild-type allele linked to a Kmr marker was inserted into the astA gene of the mutant (37) were compared to 81-176 for their ability to invade INT407 cells. The mutant invaded at <0.0015% of the input inoculum (>1,000-fold reduced compared to the wild type), as shown in Fig. 2A, and invasion was restored in the complement. For comparison, mutations were constructed in 81-176 in ciaB and flaC. As seen in Fig. 2B, both mutants were reduced in invasion compared to 81-176 but much less so than the Cj0977 mutant and less than the corresponding mutations in other strains (19, 20, 29, 31).

    We examined expression of the Cj0977 protein in 10 mutants in the flagella regulon of 81-176 as shown in Table 1. All of the mutants in Table 1 were nonmotile, except for the fliA mutant, which has a truncated flagella filament that confers reduced motility (13). Electron microscopic examination indicated that the remaining mutants were bald, except for the flaA flaB mutant and the fliD mutant, both of which produced hook structures. Representative electron micrographs are shown in Fig. 3. The Cj0977 protein could be observed in immunoblots of whole-cell preparations of wild-type 81-176 and the complement but was missing in the mutant, as seen in Fig. 4B. Figure 4 also shows that no Cj0977 protein could be detected in the fliA mutant, and Cj0977 protein levels were significantly reduced in mutants in flgR, flgS, rpoN, flgE, flhA, flhB, and fliR. The level of expression appeared to be slightly reduced in a flaA flaB double mutant, and expression appeared to be equivalent to or perhaps increased in the fliD mutant. The same preparations were also immunoblotted with anti-Omp50 (3) antibody to confirm equal loading (Fig. 4A and C).

    The Cj0977 mutant and the complemented strain were compared to wild-type 81-176 in the ferret diarrhea model (1). The Cj0977 mutant was attenuated compared to both the parent and the complement, as shown in Fig. 5. Thus, fewer animals developed diarrhea over the course of the experiment when fed the mutant (3 of 16 ferrets) compared to the wild type (12 out of 16; P = 0.0038) or complement (11 out of 16; P = 0.0113), and those animals that did develop diarrhea following feeding of the mutant displayed symptoms later than those fed the wild type or complement.

    Although we have not been able to demonstrate secretion of Cj0977 into the supernatant (data not shown), the invasion defect in the Cj0977 mutant of 81-176 was greater than mutation of either ciaB or flaC in the same strain. In TGH9011, the flaC mutant invaded at about 14% of the level of the wild type compared to 43% of the level of the wild type in 81-176. Similarly, while the ciaB mutant in F38011 invaded at levels that were about 50-fold lower than those of the parent, the attenuation in 81-176 was 72% of the wild type. The discrepancy may be due, in part, to technical differences in invasion assays and cell lines used among different laboratories. However, the observed variations may reflect inherent differences in microtubule and microfilament uptake mechanisms among strains (6, 16-18, 25, 27, 30).

    While much additional work remains to be done to understand the mechanism by which Cj0977 contributes to virulence of 81-176 at the molecular and cellular levels, these preliminary data establish a role for this protein in pathogenesis. This is the first report of coregulation of a virulence determinant with the C. jejuni flagella regulon, and it is similar to reports of 28-regulated virulence genes in other pathogens (8, 14, 15). The data also provide an additional explanation of the role of flagella in pathogenesis of C. jejuni, namely that this virulence gene, and perhaps others that are also 28 or 54 regulated (5 and Goon et al., unpublished), form an integral part of the flagella regulon.

    ACKNOWLEDGMENTS

    We thank Dave Hendrixson and Vic DiRita for the rpoN and fliA deletion mutants of 81-176, Lindsay Holder and Anahita Kiavand for technical assistance, and Robert Williams for electron microscopy.

    This work was supported by RO1 AI043559 and NMRC work unit 6000.RAD1.DA3.A0308 from the Military Infectious Diseases Research Program.

    REFERENCES

    1. Bacon, D. J., C. M. Szymanski, D. H. Burr, R. P. Silver, R. A. Alm, and P. Guerry. 2001. A phase variable capsule is involved in virulence of Campylobacter jejuni 81-176. Mol. Microbiol. 40:769-777.

    2. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M. J. Blaser. 1988. Experimental Campylobacter jejuni infections in humans. J. Infect. Dis. 157:472-479.

    3. Bolla, J. M., E. De, A. Dorez, and J. M. Pages. 2000. Purification, characterization and sequence analysis of Omp50, a new porin isolated from Campylobacter jejuni. Biochem. J. 352:637-643.

    4. Caldwell, M. B., P. Guerry, E. C. Lee, J. P. Burans, and R. I. Walker. 1985. Reversible expression of flagella in Campylobacter jejuni. Infect. Immun. 50:941-943.

    5. Carrillo, C. D., E. Taboada, J. H. E. Nash, P. H. Lanthier, J. Kelly, P. C. Lau, R. Verhulp, O. Mykytczuk, J. Sy, W. A. Findlay, K. Amoako, G. Gomis, P. Willson, J. W. Austin, A. Potters, L. Babiuk, B. Allan, and C. M. Szymanski. 2004. Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA. J. Biol. Chem. 279:20327-23008.

    6. deMelo, M. A., G. Gabbiani, and J. C. Pechere. 1989. Cellular events and intracellular survival of Campylobacter jejuni during infection of HEp-2 cells. Infect. Immun. 57:2214-2222.

    7. Doig, P., N. Kinsella, P. Guerry, and T. J. Trust. 1996. Characterization of a post-translational modification of Campylobacter flagellin: identification of a sero-specific glycosyl moiety. Mol. Microbiol. 19:379-387.

    8. Eichelberg, K., and J. E. Galan. 2000. The flagellar sigma factor FliA (28) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system. Infect. Immun. 68:2735-2743.

    9. Goon, S., J. F. Kelly, S. M. Logan, C. P. Ewing, and P. Guerry. 2003. Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene. Mol. Microbiol. 50:659-671.

    10. Grant, C. C. R., M. E. Konkel, W. Cieplak, Jr., and L. S. Tompkins. 1993. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect. Immun. 61:1764-1771.

    11. Guerry, P., R. Yao, R. A. Alm, D. H. Burr, and T. J. Trust. 1994. Systems of experimental genetics for Campylobacter species. Methods Enzymol. 235:474-481.

    12. Guerry, P., C. P. Ewing, T. E. Hickey, M. M. Prendergast, and A. P. Moran. 2000. Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni. Infect. Immun. 68:6656-6662.

    13. Hendrixson, D. R., and V. J. DiRita. 2003. Transcription of 54-dependent but not 28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus. Mol. Microbiol. 50:687-702.

    14. Heuner, K., C. Dietrich, C. Skriwan, M. Steinert, and J. Hacker. 2002. Influence of the alternate 28 factor on virulence and flagellum expression of Legionella pneumophila. Infect. Immun. 70:1604-1608.

    15. Heuner, K., and M. Steinert. 2003. The flagellum of Legionella pneumophila and its link to the expression of the virulence phenotype. Int. J. Med. Microbiol. 293:133-143.

    16. Hu, L., and D. J. Kopecko. 1999. Campylobacter jejuni associates with microtubules and dynein during invasion into human intestinal cells. Infect. Immun. 67:88-93.

    17. Konkel, M. E., S. F. Hayes, L. A. Joens, and W. Cieplak, Jr. 1992. Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures. Microb. Pathogen. 13:357-370.

    18. Konkel, M. E., and L. A. Joens. 1989. Adhesion to and invasion of HEp-2 cells by Campylobacter spp. Infect. Immun. 57:2984-2990.

    19. Konkel, M. E., B. J. Kim, V. Rivera-Amill, and S. G. Garvis. 1999. Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured epithelial cells. Mol. Microbiol. 32:691-701.

    20. Konkel, M. E., J. D. Klena, V. Rivera-Amill, M. R. Monteville, D. Biswas, B. Raphael, and J. Mickelson. 2004. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagella export apparatus. J. Bacteriol. 186:3296-3303.

    21. Labigne-Roussel, A., P. Couroux, and L. S. Tompkins. 1988. Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni. J. Bacteriol. 170:1704-1708.

    22. Lee, A., J. L. O'Rourke, P. J., Barrington, and T. J. Trust. 1986. Mucus colonization as a determinant of pathogenicity in intestinal infection by Campylobacter jejuni: a mouse cecal model. Infect. Immun. 51:536-546.

    23. Logan, S. M., J. F. Kelly, P. Thibault, C. P. Ewing, and P. Guerry. 2002. Structural heterogeneity of carbohydrate modifications affects serospecificity of campylobacter flagellins. Mol. Microbiol. 46:587-597.

    24. Morooka, T., A. Umeda, and K. Amako. 1985. Motility as an intestinal colonization factor for Campylobacter jejuni. J. Gen. Microbiol. 131:1973-1980.

    25. Monteville, M. R., J. E. Yoon, and M. E. Konkel. 2003. Maximal adherence and invasion of INT 407 cells by Campylobacter jejuni requires the CadF outer-membrane protein and microfilament reorganization. Microbiology 149:153-165.

    26. Newell, D. G., H. McBride, and J. M. Dolby. 1985. Investigations on the role of flagella in the colonization of infant mice with Campylobacter jejuni and attachment of Campylobacter jejuni to human epithelial cell lines. J. Hyg. 95:217-227.

    27. Oelschlaeger, T. A., P. Guerry, and D. J. Kopecko. 1993. Novel microtubule dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii. Proc. Natl. Acad. Sci. USA 90:6884-6888.

    28. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev, S. Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H. van Vliet, S. Whitehead, and B. G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668.

    29. Rivera-Amill, V., B. J. Kim, J. Seshu, and M. E. Konkel. 2001. Secretion of the virulence-associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal. J. Infect. Dis. 183:1607-1616.

    30. Russell, R. G., and D. C. Blake, Jr. 1994. Cell association and invasion of Caco-2 cells by Campylobacter jejuni. Infect. Immun. 62:3773-3779.

    31. Song, Y. C., S. Jin, H. Louie, D. Ng, R. Lau, Y. Zhang, R. Weerasekera, S. A. Raschid, L. A. Ward, S. D. Der, and V. L. Chan. 2004. FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagella apparatus, binds epithelial cells and influences cell invasion. Mol. Microbiol. 53:541-553.

    32. Szymanksi, C. M., M. King, M. Haardt, and G. D. Armstrong. 1995. Campylobacter jejuni motility and invasion of Caco2 cells. Infect. Immun. 63:4295-4300.

    33. Thibault, P., S. M. Logan, J. F. Kelly, J.-R. Brisson, C. P. Ewing, T. J. Trust, and P. Guerry. 2001. Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin. J. Biol. Chem. 276:34862-34870.

    34. Wassenaar, T. M., N. M. C. Bleumink-Pluym, and B. A. M. van der Zeijst. 1991. Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion. EMBO J. 10:2055-2061.

    35. Yao, R. R., A. Alm, T. J. Trust, and P. Guerry. 1993. Construction of new Campylobacter cloning vectors and a new chloramphenicol mutational cassette. Gene 130:127-130.

    36. Yao, R., D. H. Burr, P. Doig, T. J. Trust, H. Niu, and P. Guerry. 1994. Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells. Mol. Microbiol. 14:883-893.

    37. Yao, R., and P. Guerry. 1996. Molecular cloning and site-specific mutagenesis of a gene involved in arylsulfatase synthesis in Campylobacter jejuni. J. Bacteriol. 178:3335-3338.(Scarlett Goon, Cheryl P. )